Chemokines: structure, receptors and functions. A new target for inflammation and asthma therapy?

Five to 10% of the human population have a disorder of the respiratory tract called ‘asthma’. It has been known as a potentially dangerous disease for over 2000 years, as it was already described by Hippocrates and recognized as a disease entity by Egyptian and Hebrew physicians. At the beginning of this decade, there has been a fundamental change in asthma management. The emphasis has shifted from symptom relief with bronchodilator therapies (e.g. β2-agonists) to a much earlier introduction of anti-inflammatory treatment (e.g. corticosteroids). Asthma is now recognized to be a chronic inflammatory disease of the airways, involving various inflammatory cells and their mediators. Although asthma has been the subject of many investigations, the exact role of the different inflammatory cells has not been elucidated completely. Many suggestions have been made and several cells have been implicated in the pathogenesis of asthma, such as the eosinophils, the mast cells, the basophils and the lymphocytes. To date, however, the relative importance of these cells is not completely understood. The cell type predominantly found in the asthmatic lung is the eosinophil and the recruitment of these eosinophils can be seen as a characteristic of asthma. In recent years much attention is given to the role of the newly identified chemokines in asthma pathology. Chemokines are structurally and functionally related 8–10 kDa peptides that are the products of distinct genes clustered on human chromosomes 4 and 17 and can be found at sites of inflammation. They form a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and lymphocytes. The chemokine superfamily can be divided into three subgroups based on overall sequence homology. Although the chemokines have highly conserved amino acid sequences, each of the chemokines binds to and induces the chemotaxis of particular classes of white blood cells. Certain chemokines stimulate the recruitment of multiple cell types including monocytes, lymphocytes, basophils, and eosinophils, which are important cells in asthma. Intervention in this process, by the development of chemokine antagonists, might be the key to new therapy. In this review we present an overview of recent developments in the field of chemokines and their role in inflammations as reported in literature

Characterization of endothelial cells of lymphatic vessels

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice only tumours of the blood vessel endothelium (haemangiomas) have been thus far reported. In the first part of this thesis is described a highly reproducible method for the induction of benign tumours of the lymphatic endothelial cells (lymphangioma) in mice, by intraperitoneal injection of incomplete Freund's adjuvant. Different criteria have been used in order to establish the nature of the induced lesion. Morphological and histophatological studies of the tumour developed in the peritoneal cavity revealed the presence of cells at various levels of vascular development. Expression of the endothelial markers PECAM/CD31, ICAM-l/CD54, ICAM 2/CDI02 as well as the vascular endothelial growth factor (VEGF) receptor Flk-I, the endothelial cell specific receptors Tie-I, Tie-2, and the lymphatic endothelial specific Flt-4 was identified. When the lesion was induced in ~- galactosidase knock-in Flt-4+/- mice, the tumour endothelia could be stained blue in a number of tumour cells. Tumour-derived cells were propagated in vitro where they spontaneously differentiated, forming vessel-like structures. This evidence leads to the conclusion that this is the first experimental protocol for the induction of a lymphatic endothelium hyperplasia in mice peritoneum. The second part of this thesis describes the use of this model system to investigate the profile of chemokine expression in murine lymphangiomas and in lymphangioma-derived lymphatic endothelial primary cultures. Chemokines are a superfamily of small, secreted chemoattractant molecules that plays a key role in the immune cell trafficking. Although production of chemokines by vascular endothelial cells has been extensively documented, there is much less information regarding the lymphatic endothelium. The reported results are the first detailed analysis of chemokine production by lymphatic endothelial cells. Chemokines belonging to all three subfamilies (CXC, CC and C), were found to be expressed in lymphangioma. Among these molecules is remarkable the identification of CIO, a molecule previously identified only in the bone marrow. The molecular as well as functional assays performed provide an indication of the signals that mediate the recruitment of leukocytes into lymphatic vessels

Mechanisms of eosinophil adhesion to endothelial cells under flow conditions

Eosinophils play an important role in allergic inflammatory diseases such as allergic asthma. Infiltrates of these cells are present in the interstitium and the lumen of the bronchi of asthmatic patients. Eosinophils must pass the endothelium to enter this site of inflammation. A widely accepted paradigm for leukocyte extravasation is the multistep model. In this model selectins mediate rolling interactions between leukocytes and endothelium and subsequently, activated integrins facilitate firm adhesion and extravasation of the cells. The interactions between eosinophils and activated endothelial cells have been studied under flow conditions. This has been done in a flow chamber model in which a blood vessel is simulated. In chapter 1 (general introduction) the multistep model of extravasation is explained. Furthermore, eosinophils function and their role in allergic asthma has been described. In chapter 2 data are presented on the function of alpha 4 integrins and E-selectin in the initial attachment of eosinophils to TNF-? activated endothelial cells. In chapter 3 data are presented on the differences in adhesion behavior between eosinophils of healthy blood donors and of allergic asthmatic patients on activated endothelial cells under flow conditions. Remarkably, eosinophils of allergic asthmatic patients bound platelets in contrast to cells of healthy controls. The platelet-eosinophil interactions resulted in increased adhesion to activated endothelial cells via a process that is called secondary tethering. In chapter 4 data are presented on the role of IL-8 on eosinophil arrest. IL-8 is a typical neutrophil activator but is not known to be a eosinophil activator. We showed that IL-8 can induce 1) an alpha 4- and beta 2-integrin dependent arrest of rolling eosinophils on activated endothelial cells and 2) an increase in intracellular Ca2+ concentration of eosinophils binding to fibronectin and activated endothelial cells. Thus, IL-8 is not only a neutrophil activator but also an eosinophil activator. In chapter 5 data are presented on the role of phospholipase C (PLC) in the chemokine-induced integrin activation of eosinophils. Three chemokines, IL-8, eotaxin and C5a were tested on their ability to induce a PLC dependent arrest of eosinophils. Eotaxin mediated a totally PLC-dependent arrest whereas IL-8 and C5a only induced a partial PLC-dependent arrest. In resting eosinophils PLC is also important because inhibition of PLC decreased the functionality of alpha 4 integrins. In chapter 6 data are presented on the migration behavior of eosinophils. Migrating cells display a typical tear-drop shape while migrating. The front is a broad protrusion (lamellipodium) whereas the back of the cell is a small point-like structure (uropod). The small GTP-ase RhoA was found to play an important role in the regulation of the movement of the uropod. In chapter 7 (general discussion) the data of chapter 2-6 are discussed. Special emphasis was give to the validation of the model, the possible working mechanisms of PLC in integrin regulation, the role of platelets in the binding of eosinophils of allergic asthmatic patients to activated endothelial cells and possible target molecules for therapy

Development and characterization of humanized and human forms of ELR-CXC chemokine antagonist, bovine CXCL8(3-74)K11R/G31P

Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5’ half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia. As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. In vitro, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. In vivo, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS

Concepts of GPCR-controlled navigation in the immune system

G-protein-coupled receptor (GPCR) signaling is essential for the spatiotemporal control of leukocyte dynamics during immune responses. For efficient navigation through mammalian tissues, most leukocyte types express more than one GPCR on their surface and sense a wide range of chemokines and chemoattractants, leading to basic forms of leukocyte movement (chemokinesis, haptokinesis, chemotaxis, haptotaxis, and chemorepulsion). How leukocytes integrate multiple GPCR signals and make directional decisions in lymphoid and inflamed tissues is still subject of intense research. Many of our concepts on GPCR-controlled leukocyte navigation in the presence of multiple GPCR signals derive from in vitro chemotaxis studies and lower vertebrates. In this review, we refer to these concepts and critically contemplate their relevance for the directional movement of several leukocyte subsets (neutrophils, T cells, and dendritic cells) in the complexity of mouse tissues. We discuss how leukocyte navigation can be regulated at the level of only a single GPCR (surface expression, competitive antagonism, oligomerization, homologous desensitization, and receptor internalization) or multiple GPCRs (synergy, hierarchical and non-hierarchical competition, sequential signaling, heterologous desensitization, and agonist scavenging). In particular, we will highlight recent advances in understanding GPCR-controlled leukocyte navigation by intravital microscopy of immune cells in mice

Osteoid cell-derived chemokines drive bone-metastatic prostate cancer

One of the greatest challenges in improving prostate cancer (PCa) survival is in designing new therapies to effectively target bone metastases. PCa regulation of the bone environment has been well characterized; however, bone-targeted therapies have little impact on patient survival, demonstrating a need for understanding the complexities of the tumor-bone environment. Many factors contribute to creating a favorable microenvironment for prostate tumors in bone, including cell signaling proteins produced by osteoid cells. Specifically, there has been extensive evidence from both past and recent studies that emphasize the importance of chemokine signaling in promoting PCa progression in the bone environment. Chemokine-focused strategies present promising therapeutic options for treating bone metastasis. These signaling pathways are complex, with many being produced by (and exerting effects on) a plethora of different cell types, including stromal and tumor cells of the prostate tumor-bone microenvironment. ​This review highlights an underappreciated molecular family that should be interrogated for treatment of bone metastatic prostate cancer (BM-PCa)

Streptococcus pyogenes protease SpyCEP impairs neutrophil signalling

The enzyme Streptococcus pyogenes Cell Envelope Protease (SpyCEP) is critical to the pathogenesis of necrotizing fasciitis. SpyCEP is known to cleave the C-terminus of ELR+ CXC chemokines, notably the neutrophil attractant CXCL8 which binds to the cell-surface receptors CXCR1 and CXCR2. Cleavage of CXCL8 leaves the N-terminus and disulphide bonds of the chemokine intact, but removes a small portion of an α-helix known to be important in chemokine binding to glycosoaminoglycans (GAGs) on the surface of cells. The work presented in this thesis examined the interaction between SpyCEP-cleaved CXCL8 and its receptors CXCR1 and CXCR2, considered the role of glycosaminoglycan binding in the receptor:chemokine interaction and probed the impact of SpyCEP upon neutrophil death. In vitro studies in this thesis demonstrated that the chemotaxis of CXCR1+ and CXCR2+ cells towards a source of CXCL8 was impaired by SpyCEP cleavage of the chemokine. Similarly, SpyCEP-cleaved CXCL8 was found to have markedly reduced affinity for both CXCR1 and CXCR2 in ligand binding assays. Thus, SpyCEP-cleavage of CXCL8 prevents an effective interaction with its receptors and subsequent downstream signalling. SpyCEP-cleavage of CXCL8 was also found to impair the interaction of CXCL8 with GAGs, with cleaved CXCL8 unable to form multimers on heparin-coated beads. This leads us to hypothesize that SpyCEP functions in vivo by impeding the ability of CXCL8 to bind to GAGs present on the neutrophil surface, leading to a loss of receptor binding and a lack of neutrophil recruitment to the site of infection. Neutrophil survival in vitro and the impact of SpyCEP was found to be highly context-dependent, with the magnitude of difference due to SpyCEP varying markedly between culture conditions. Exposure of neutrophils to high concentrations of affinity-purified SpyCEP appeared to be associated with increased neutrophil necrosis, whilst exposure to supernatant from SpyCEP+ S.pyogenes cultures increased neutrophil death compared to SpyCEP- control supernatant. Exposure of neutrophils to live SpyCEP+ S.pyogenes may afford marginal protection to neutrophils from necrotic death induced by live S.pyogenes. Antibiotic killed bacteria did not appear to influence neutrophil survival regardless of the presence of SpyCEP. Thus, in the presence of intact bacteria, SpyCEP appears not to be a major determinant of neutrophil survival or death. Evaluation of clinical samples taken from different sites and at different timepoints from a patient with S.pyogenes necrotising fasciitis demonstrated the presence of SpyCEP in infected tissue. A range of SpyCEP concentrations within the tissue were detected, which were comparable to the SpyCEP concentrations used in the in vitro assays of neutrophil survival. From the work performed here, a greater understanding of the mechanisms by which CXCL8 interacts with its receptors has been gained, notably highlighting the importance of the chemokine C-terminal α-helix in GAG-mediated presentation of CXCL8 to its receptors. The impact of SpyCEP activity upon CXCR1 and CXCR2 signalling is more clearly understood, whilst the complexity and context-dependent nature of the relationship between CXCL8, SpyCEP, S.pyogenes and neutrophil death is better appreciated.Open Acces

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

<p>Abstract</p> <p>Background</p> <p>Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β₂-integrin activation and function in neutrophilic transmigration through endothelium.</p> <p>Methods and results</p> <p>Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β₂-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils <it>in vitro</it>. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β₂-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.</p> <p>Conclusion</p> <p>This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β₂-integrins on the cell surface. Blocking this activation of β₂-integrins might be an important target in cigarette smoke induced neutrophilic diseases.</p

Identification Of New Molecules Involved In Dendritic Cell Localization

The present study was designed to investigate the expression and function of an orphan seven transmembrane receptor named CCRL2 (CC chemokine receptor like 2), a putative chemokine receptor. In human tissues CCRL2 was expressed mainly by lung, lymphoid tissues and fetal spleen. Among leukocytes CCRL2 was expressed by monocytes, neutrophils and dendritic cells (DC). Because chemokines play a fundamental role in DC trafficking, modulation of CCLR2 expression in this cell type was further investigated. Maturative stimuli like LPS and CD40L strongly up regulated CCRL2 mRNA and protein in DC. Culture of DC in the presence of inhibitors of maturation and function such as VitD3 and Dex had no effect on LPS-induced CCRL2 up regulation. On the contrary PGE2, that does not affect DC maturation, completely abolished LPS induction of CCRL2 expression. The effect of LPS and CD40L on CCRL2 expression was rapid (1.5h) and transient (maximal at 4h) and declined by 24h, conversely the upregulation of CCR7 that was slower and reached a plateau at 24h of stimulation. Since CCRL2 gene is located in the main chemokine receptor cluster in the 3p21 chromosome, it is likely to be a conventional inflammatory chemokine receptor. In order to identify ligands CCRL2 transfectants were used in chemotaxis and calcium flux assays with a broad panel of inflammatory CC and CXC chemokines but no ligand was identified. The alterations in the DRYLAR/IV motif in the second intracellular loop suggest that CCRL2 may be a candidate for the family of chemokine decoy receptors like the receptor D6. This second hypothesis was evaluated performing chemokine scavenging assays. None of the chemokine tested was scavenged by CCRL2. However in parallel experiments two new ligands for D6, the CCR4 agonists CCL17 and CCL22 were identified. In summary these data suggest that CCRL2 might be involved in DC trafficking, through the regulation of the DC emigration from tissues following stimulation. None of the chemokine tested was able to bind or activate CCRL2. Furthermore CCRL2 appears not to act as a chemokine scavenger receptor and its biological role is still elusive